Flavylium dye as pH-tunable fluorescent and CD probe for double-stranded DNA and RNA

UIDB/50006/2020 ROTEIRO/0031/2013-PINFRA/22161/2016 HrZZ IP-2018-01-5475The interaction of 4′-(N,N-dimethylamino)-6-hydroxyflavylium cation with double stranded (ds-) DNA/RNA was studied by UV/Vis spectrophotometry, circular dichroism (CD), and also steady-state and time-resolved emission spectroscopies at neutral and weakly acidic conditions. At pH 5, the studied molecule, in its flavylium cationic form, showed considerable binding affinities (5 < logKs < 6) for all ds-DNA/RNA, contrary to chalcones forms (dominant at pH 7), which did not show binding to polynucleotides. Flavylium cation intercalated into ds-DNAs at variance to dominant groove aggregation within ds-RNA, which was reported by RNA-specific bisignate induced CD spectrum (ICD) bands. The intrinsically negligible fluorescence of flavylium was strongly increased upon the addition of DNA or RNA, whereby both the fluorescence intensity and emission lifetimes of complexes differed considerably: the strongest emission increase was observed for AU-RNA (detection limit estimated to 10 nM) followed by AT-DNAs and the much weaker effect of GC-DNAs. Both fluorescence sensitivity on the ds-DNA/RNA secondary structure and sequence-selective ICD bands make the flavylium–chalcones system an intriguing pH-switchable new probe for distinguishing between various polynucleotide sequences.publishersversionpublishe

Come-back of phenanthridine and phenanthridinium derivatives in 21st century

Phenanthridine derivatives are one of the most intensively studied families of biologically active compounds with efficient DNA binding ; attracting attention about time of DNA structure discovery (1960ies), early recognized as a symbol of DNA intercalative binding, for many decades applied as gold-standard DNA- and RNA-fluorescent markers (ethidium bromide), probes for cell viability (propidium iodide), but also “ill-famed” for various toxic (genotoxic) and mutagenic effects. After two decades of low interest, the discovery of phenanthridine alkaloids and new studies of antiparasitic/antitumour properties of phenanthridine derivatives resulted in the strong increase of the scientific interest about the turn of this century. Here are summarized phenanthridine-related advances in the 21st century (2000-present period) with emphasis on the supramolecular interactions and bioorganic chemistry, as well as novel or improved synthetic approaches

The Phenanthridine-modified Tyrosine Dipeptide: Synthesis and Non-covalent Binding to DNA and RNA

Dipeptide 4 containing two unnatural amino acids, a modified tyrosine and a phenanthridine derivative, was synthesized. Binding of the dipeptide to a series of polynucleotides including ct-DNA, poly A - poly U, poly (dAdT)2, poly dG - poly dC and poly (dGdC)2 was investigated by thermal denaturation experiments, fluorescence spectroscopy and circular dichroism. Thermal denaturation experiments indicated that dipeptide 4 at pH 5.0, when phenanthridine is protonated, stabilizes ds-DNA, whereas it destabilizes ds-RNA. At pH 7.0, when the phenanthridine is not protonated, effects of 4 to the polynucleotide melting temperatures are negligible. At pH 5.0, dipeptide 4 stabilized DNA double helices, and the changes in the CD spectra suggest different modes of binding to ds-DNA, most likely the intercalation to poly dG- poly dC and non-specific binding in grooves of other DNA polynucleotides. At variance to ds-DNA, addition of 4 destabilized ds-RNA against thermal denaturation and CD results suggest that addition of 4 probably induced dissociation of ds-RNA into ss-RNA strands due to preferred binding to ss-RNA. Thus, 4 is among very rare small molecules that stabilize ds-DNA but destabilize ds-RNA. However, fluorescence titrations with all polynucleotides at both pH values gave similar binding affinity (log Ka ≈ 5), indicating nonselective binding. Preliminary photochemical experiments suggest that dipeptide 4 reacts in the photochemical reaction, which affects polynucleotides chirality, presumably via quinone methide intermediates that alkylate DNA. This work is licensed under a Creative Commons Attribution 4.0 International License

Great Efficiency of Nucleosides as Organizing Tools

Bis-porphyrins with flexible linkers such as uridine or 2’-deoxyuridin pre-organize in a face-to-face conformation and form stable sandwich complexes with bidentate base such as DABCO. The gain in stability can be even greater when a dinucleotide linker is used. Such pre-organization increases the association constant by one to two orders of magnitude when compared to the one of DABCO with a reference porphyrin. Comparison with rigid tweezers shows a better efficiency of nucleosidic dimers. The choice of rigid spacers is not the only way to pre-organize bis-porphyrins, and well-chosen nucleosidic linkers offer an interesting option for the synthesis of such devices

2,3-Dimethylbenzoxazolium Methosulfate

An economically benign solvent-free approach to synthesise 2, 3-dimethylbenzoxazolium methosulfate is reported in the present work. The title compound is derived from 2-methylbenzoxazole reacting with a slight excess of dimethylsulfate, at room temperature. The reaction proceeds via an intrinsic exothermic reaction, and the benzoxazolium salt crystallized after a short time into a white crystalline form. The product was filtered off and washed with acetone and diethyl ether to provide the desired product in 89% yield. The target compound was evaluated by ESI/MS analysis

Interactions of variously coated gold and silver nanoparticles with a bis(triarylborane) photodyanmic therapy (PDT)-dye; their cellular uptake, cytotoxicity and photo-activity

Background and purpose: Diethynylarene-linked bis(triarylborane) tetracations can be used as probes for fluorimetric and Raman sensing of biomacromolecules, as well as promising theragnostic agents. Among them, bis(triarylborane) fluorophore (TAB3), when bonded to Ag nanoparticles (NP), stood out with specific properties such as Raman signal enhancemen of the TAB3 dye in a cuvette. However, TAB3 dye - nanoparticle composites have not been studied in biological systems. For this reason, questions arose as to whether different types of metal nanoparticles (Au or Ag-based) with different coatings (negatively charged citrate or neutral PVP) could be efficiently stained with the TAB3 dye in a cuvette. The aim of this research was to examine Au and Ag nanoparticles of similar size (20-25 nm) with different stabilizers for their cellular uptake, cytotoxicity in the dark and under visible light radiation, to characterize the interactions of nanoparticles with the TAB3 fluorophore, and to study NP-TAB3 composites in cells, evaluate their intracellular staining, as well as possible photoinduced release and biological activity. Materials and methods: The binding constants of Au- and Ag- based nanoparticles with TAB3 were determined by fluorimetric titrations. The cytotoxic effect of NPs was determined by the survival of A549 cells (MTT assay). Cellular uptake of both NP and NP-TAB3 composites were performed by live cell imaging experiments. Results: The Au- or Ag-based NPs with different coatings bind to the TAB3 with high affinity. These NPs, as well as TAB3-NP complexes, efficiently enter living human cells, accumulating in cytoplasm with no apparent selectivity for a particular organelle. Even prolonged 3-day treatment with the NPs studied did not show any toxic effect on the cells. Bioimaging studies in cells revealed that the TAB3-NP complex does not intracellularly dissociate; the previously reported photo-bioactivity of TAB3 is completely inhibited by binding to NPs. Conclusion: Au- and Ag NPs were non-covalently stained by TAB3, irrespective of the different coatings, with similar binding affinities. Emission from TAB3 is strongly quenched by the NPs, but not completely. Experiments on living human cells revealed that neither free NPs, nor their composites with TAB3, were toxic. Bioimaging studies by confocal microscopy revealed that all NPs efficiently enter living cells within 90 min. Colocalization experiment with simultaneous collection of data in the reflection and fluorescence modes demonstrated that the TAB3 dye remained bound to NPs inside cells. Strong irradiation of TAB3-NP inside cells with a 457 nm laser did not yield any damage to the cells, at variance with our previously shown very strong photo-bioactivity of the TAB3 dye alone. Thus, binding of a chromophore to a nanoparticle can inhibit the chromophore’s ability to undergo photo-induced singlet oxygen production, consequently blocking its photo-bioactivity